FR2829151A1 - PROCESS FOR OBTAINING PROCESSED CORN PLANTS WITH IMPROVED DIGESTIBILITY CHARACTERISTICS, CORN PLANTS OBTAINED BY THE PROCESS AND USES - Google Patents

Abstract

The present invention relates to the use of a polynucleotide modulating the synthesis of corn methyltransferase COMT encoded by the nucleotide sequence SEQ ID No. 1, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between the S and G units of the modified lignin and having a modified lignin content remaining compatible with the normal development of the plant. Application to improving the digestibility of corn

Description

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 The present invention relates to the field of improving corn digestibility characteristics by modifying the composition and content of lignins.

PRIOR ART
Ensiled corn is an interesting food from many points of view.

For the farmer, the field yield of maize is relatively high, harvesting and storage are easy. For animals, especially for dairy cows, the nutritional qualities of ensiled corn are stable and can easily be supplemented with protein by other forage silage or soybean meal. These nutritional qualities allow the persistence of milk production. Thus, a mixture of corn silage and fodder silage makes it possible to valorize the use of fodder proteins whose solubility (50%) is very high and very fast in the rumen of the cow (Léonard, 1996). . The low calcium and potassium content of corn silage dilutes the high content of these elements in forage silage, particularly legume silage, and achieves a near perfect balance between energy and proteins from the beginning to the end of lactation. Silage is often an element that helps the farmer to master energy-protein synchrony. Given the nutritional qualities of silage maize, the cultivation of this type of forage is widespread and covers more than 3 million hectares in the European Union. Optimizing the quality of corn silage involves increasing the net energy provided by this type of feed by improving its digestibility.

 An important factor in decreasing digestibility of maize forage is related to the presence in the walls of plant cells of phenolic compounds, in particular lignins. Lignins establish different types of binding with other parietal constituents to form a tight mesh that impedes the accessibility of carbohydrates to digestive enzymes, the main source of energy for herbivores. Depending on their stage of maturity, the plants do not have the same type of lignin deposited on their cell walls. The increase in lignification during plant maturation causes a decrease in the digestibility of this plant (Bentley, 1959 and Grabber et al., 1992). It is difficult to correlate the reduction

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 of the digestibility of plants as they ripen with lignin content, lignin composition, lignin structure, or with any other parietal component given the changes in chemical constituents and physical structure associated with ripening of the plant. plant.

 Therefore, one of the preferred ways of improving the quality of silage corn is to change the composition and lignin content of forage plants. Indeed, a small decrease in the lignin content leads to a strong improvement in digestibility. An experiment conducted by Emile (1995) shows that a more digestible variety can increase the level of milk production by more than one kilogram and the weight gain of cows by 22 kg compared to a less digestible variety. Thus, the more digestible the variety, the higher the milk production and the lower the loss of flesh condition.

 Brown midrib maize, especially bm3 maize, has a greatly increased digestibility compared to conventional maize and significantly improves milk production. These bm3 maize differ from normal maize by a greatly reduced lignin content (up to 40%) and modified S units (sinapyl alcohol) / G unit (coniferyl alcohol) ratios. However, the disadvantages of corn bm3 such as a lower field yield, an increased susceptibility to lodging, a lack of growth and vigor at the beginning of vegetation and a delay in flowering prevent its exploitation (Barrière and Argillier ( 1993)).

 Lignin is a major component of the cell wall of sclerenchyma and xylem of vascular plants. It plays an important role in the conductive functions of the xylem by reducing the water permeability of the cell walls. In lignified tissues, it acts as an intercellular binding agent. It is responsible for the rigidity of the cell walls and the upright growth of plants and contributes to the resistance of plants to mechanical aggression (wind, injury ...) or infection by pathogens, tolerance to heat and water stress.

In general, it is considered that lignin is an insoluble polymer of
3 monomers of alcohols or monolignols: p-coumaryl alcohol (H units), coniferyl alcohol (G units) and sinapyl alcohol (S units). Each type of precursor can form a variety of bonds with other precursors and thus constitute lignin. Other connections may also be established

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 with other parietal compounds (polysaccharides and proteins) to form a complex three-dimensional network. The content and composition of lignins vary between large groups of higher plants and between species. The two major classes are gymnosperm lignins which contain mainly G units and a small proportion of H units, and angiosperm lignins which contain both S, G units and a small proportion of H units (Whetten et al. al., 1998). Monocotyledonines lignins, and more particularly cereals are the most complex. They consist of 3 units H, G, S to which are added in significant proportion of hydroxycinnamic acids (ferulic acid and coumaric) linked by ether bonds or esters. This structural heterogeneity is also found within the same plant, depending on the organ considered, but also depending on the stage of development and localization in the wall (Lewis and Yamamoto, 1990).

 The lignin biosynthetic pathway is shown in Figure 1.

Although the biosynthesis pathway of lignins is much studied, many steps are still misunderstood including those involved in methoxylation.

The current hypothesis of the monolignol biosynthesis pathway considers that the metabolic network leading to the formation of S and G units involves successive reactions of hydroxylation and O-methylation. The phenylpropanoides biosynthesis pathway begins with the conversion of phenylalanine to cinnamic acid under the action of
Phenylalanine-Ammonia-Lyase (PAL). The product of the PAL activity on phenylalanine is trans-cinnamic acid which is a substrate of cinnamate-4-hydroxylase (C4H), a P450-hydroxylase. C4H hydroxylates the 4-position of cinnamate to produce p-coumaric acid. In monocotyledons, p-coumaric acid results from the action of Tyrosine
Ammonia-Lyase (TAL) on tyrosine.

All of the enzymes involved later in the metabolic network have been reviewed (Boudet et al., 1995, Dixon et al., 2001;
Whetten et al., 1998, Li et al., 2000), they include: O-methyltransferases with distinct designations according to the species: caffeic acid 3-O-methyltransferase (COMT)

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also known as caffeic acid O-methyltransferase (COMT), O-methyltransferase COMT or, preferably, in the text methyltransferase COMT or 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT) and the Caffeine coenzyme A 3-0-methyltransferase (CCOAMT) , hydroxycinnamate coenzyme A ligases (4CL), ferulose 5-hydroxylase (F5H) cytochrome P450, p-coumarate hydroxylase (C3H), and several isoforms of Cinnamyl Co A reductase (CCR) and
Cinnamyl alcohol dehydrogenase (CAD).

 The final stage of polymerization of monolignols probably involves parietal enzymes, peroxidases and / or laccases.

 The enzymes catalyzing the methylation steps are O-methyltransferases. O-methyltransferases (S-adenoxyl-L-methionine O-methyltransferases, EC 2.1.1.6) play an important role in the biosynthesis of monolignols. It is currently accepted that COMT would intervene preferentially in the synthesis of S units and CCoAOMT in the synthesis of G units.

3-hydroxylase)/CCoAOMT, avec la 4CL, la CCR et la CAD sont associés à la synthèse des unités G, et la CAld5H (Coniféraldéhyde 5- hydroxylase)/AldOMT se connecte à partir du conyféryl aldéhyde pour la biosynthèse des unités S. L'implication des COMT dans la biosynthèse des unités S de lignine est supportée par une forte réduction en unité S de la lignine mais pas des unités G dans les plantes transgéniques sous-exprimant COMT would allow the methylation of free forms of hydroxycinnamic acids: caffeic acid and 5-hydroxyferulic acid by introducing one or two methoxy groups respectively into the lignin monomers (Davin and Lewis, 1992). Recently, it has been shown in poplar that COMT is actually a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT). Indeed, 5-hydroxyconiferaldehyde is the preferred substrate of AldOMT. Thus, for Li et al. (2000), there are two distinct hydroxylation / methylation pathways in woody angiosperms leading to the S and G units respectively: CCoA3H (Coumaroyl CoA

3-hydroxylase) / CCoAOMT, with 4CL, CCR and CAD are associated with the synthesis of G units, and CAld5H (Coniferaldehyde 5-hydroxylase) / AldOMT connects from conyferyl aldehyde for the biosynthesis of S units. The involvement of COMTs in the biosynthesis of lignin S units is supported by a large reduction in lignin S units but not G units in transgenic plants under-expressing

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la COMT comme le tabac (Atanassova et al., 1995), la luzerne (Guo et al., 2001) et le peuplier (Van Doorselaere et a/., 1995).

COMT such as tobacco (Atanassova et al., 1995), alfalfa (Guo et al., 2001) and poplar (Van Doorselaere et al., 1995).

 The S and G units are therefore important components of lignins and therefore play a role in the digestibility of the cell wall. However, the impact on digestibility of the increase or decrease of S and / or G units varies across studies. Thus, studies on the mutant bm3 have shown that the increased digestibility of the walls is associated with a strong alteration of the lignin structure: low S / G ratio with substantial incorporation of 5-hydroxyguaiacyl (5-OH G) units (Lapierre and a /., 1988) and a lower lignin content. If the correlation results between the digestibility and the lignin content of the plant are always negative, the correlation results between the S / G ratio and the lignin content and the digestibility are not always identical. Thus, for Zhong et al. (1998) and Mechin et al. (2000), the S / G ratio is positively correlated with digestibility. In other words, the respective results of these authors on tobacco and maize show that the higher the S / G ratio, the higher the digestibility. Thus, depending on the species and the studies, it appears that a variation of the S / G ratio, associated or not with a reduced lignin content can be accompanied by a stability, a reduction or an increase of the digestibility.

 These contradictory results demonstrate that it is not obvious to predict the consequences of the modification of the O-methyltransferase activity (COMT, AldOMT) on the S / G ratio and the lignin content and consequently on the digestibility of the plant. The invention described in this patent makes it possible to obtain maize plants offering better digestibility by modifying the O-methyltransferase activities.

 In particular, those skilled in the art are not able to transpose the highly contradictory experimental results described in the state of the art relating to the consequences of the overexpression or, conversely, of the under-expression of an enzyme involved in the biosynthesis of lignin in a given plant species, including maize, on the content and composition of lignin, in particular on the ratio of S units to G units.

 Moreover, one skilled in the art, in the light of the experimental results described in the state of the art, could not predict, in the specific case

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 maize, if an increase or decrease in the ratio of S units / units of lignin and / or lignin content would positively or negatively affect the digestibility characteristics of that plant species or the corresponding transformation products, eg forage produced from corn.

SUMMARY OF THE INVENTION
It has been shown according to the invention that, surprisingly, the induction of the modulation of the synthesis of an enzyme involved in the corn lignin biosynthesis pathway, the caffeic acid 3-0 Methyltransferase, also designated COMT, hereinafter referred to as the COMT methyltransferase text, caused a change in the ratio of S units / G units in the composition of lignin and / or lignin content, compared to that found in wild corn, and that the modification of the S / G unit ratio and / or the lignin content conferred improved characteristics of digestibility on the maize plant and its transformation products.

 By modulation of the synthesis of an enzyme is meant according to the invention a decrease, or on the contrary an increase in the synthesis of said enzyme.

 By modifying the ratio of units S / G units in the composition of lignin means a decrease or an increase in this ratio.

Wild maize means a maize with digestibility characteristics, a ratio of S units to lignin G units, and a lignin content in the plant similar to, or identical to, that of the maize line. designated WT in the examples, which is a line available from the National Institute of
Agronomic Research (INRA) under the name12.

 According to the invention, the improved digestibility characteristics of corn, or of these derived products, are obtained by modulating the synthesis of the methyltransferase COMT, which synthesis modulation induces: (i) the modification of the ratio of units S / units G in the composition of lignin; and or

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 (ii) the modification of the lignin content in the maize plant and the products derived therefrom.

 According to the invention, the modulation of the synthesis of the methyltransferase COMT can be completed by the simultaneous modulation of the synthesis of a second enzyme involved in the lignin biosynthesis pathway, preferably a second enzyme involved in the pathway of biosynthesis of lignin in maize as described in the prior art and in particular illustrated in the diagram of FIG.

 The decrease in COMT methyltransferase synthesis can be achieved by transforming maize plants, especially wild maize plants, with an antisense polynucleotide inhibiting the translation of the corresponding messenger RNA. This reduction can also be obtained by a co-suppression phenomenon, as described for example by Napoli et al. (1990) or Carvalho Niebel et al. (1995).

 Conversely, the increase in the synthesis of the methyltransferase COMT can be obtained by transforming maize plants, in particular wild corn plants, with a polynucleotide encoding this enzyme.

 Whatever the polynucleotide used, it preferably comprises at least one regulatory sequence allowing its expression in a corn cell.

 According to the invention, a reduction of the ratio of units S / units G is obtained at least by specific inhibition of the COMT activity. It can be supplemented by the increase or reduction of the synthesis of other enzymes involved in the biosynthesis of S units and G units of maize lignin, an increase or reduction of activity obtained mainly by overexpression or, conversely, inhibition of the expression of genes encoding these other enzymes.

 An inhibition of the corn COMT activity is mainly obtained according to the invention by the use of oligonucleotides specifically inhibiting the transcription or translation of the gene encoding this enzyme.

 The nucleic acid encoding corn COMT is referenced as SEQ ID? 1 of the sequence listing.

 The subject of the invention is the use of a polynucleotide modulating the synthesis of corn comt methyltransferase encoded by the sequence

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 nucleotide SEQ ID N'l, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between units S and G of the modified lignin and / or having a modified lignin content remaining compatible with the normal development of the plant.

 It also relates to a process for obtaining corn plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or whose lignin content is modified and compatible with the normal development. of the plant, said method comprising a step of transforming a maize host cell with a polynucleotide selected from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding corn COMT methyltransferase; b) a polynucleotide encoding corn COMT methyltransferase.

 Preferably, the antisense polynucleotide is the polynucleotide of sequence SEQ ID. 2.

 Preferably, the polynucleotide encoding corn COMT methyltransferase is the polynucleotide of sequence SEQ ID No. 1.

 The antisense polynucleotide or the polynucleotide encoding corn COMT methyltransferase are preferably inserted into an expression cassette allowing their functional expression in corn.

 Such an expression cassette is advantageously introduced into an expression vector adapted to transform corn cells.

 According to the method of the invention, it is possible to use a second polynucleotide modulating the activity of an enzyme involved in the biosynthetic pathway of the S units and lignin G units, other than the COMT methyltransferase.

 The invention also relates to a maize plant transformed according to the above method, which has improved digestibility characteristics, as well as to any part of the transformed maize plant, in particular root, seed, leaf, flower and stem.

 It also relates to the transformation products obtained from the transformed maize plants defined above, in particular fodder and purified lignin.

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DESCRIPTION OF THE FIGURES FIG. 1: Biosynthetic pathway of lignin monomers (Guo et al., 2001).

 The "metabolic network" described in the scheme includes the results of recent studies suggesting the unexpected substrate specificities of the F5H and COMT enzymes (Humphreys et al 1999, Osakabe et al 1999, Li et al 2000). The framed pathway (guaiacyl [G] unit) represents the main reactions leading to lignin G. The framed path (syringyl [8] unit) represents the preferred biosynthetic pathway for lignin S, 4C1, 4coumarate coenzyme A ligase; CAD, cinnamyl alcohol dehydrogenase; CCR, cinnamyl coenzyme A reductase.

transformation visant l'activité COMT sur la structure des lignines. Les unités H (R1=R2=H), G (R1=OCH3, R2=H), S (R1=R2=OCH3) et 5-OH G (R1= OCH3, R2=OH) engagées dans des liaisons-0-4 génèrent des monomères thioéthylés spécifiques. Après silylation (BSTFA), ces monomères sont analysés en CPG-SM (impact électronique). Les chromatogrammes reconstruits sur les ions benzyliques propres à chacun d'eux permettent d'évaluer de manière univoque et sans interférence des autres constituants la fréquence relative des unités ayant généré ces monomères. Figure 2: Principle of the method used to assess the impact of the

transformation targeting COMT activity on lignin structure. The units H (R1 = R2 = H), G (R1 = OCH3, R2 = H), S (R1 = R2 = OCH3) and 5-OH G (R1 = OCH3, R2 = OH) engaged in 0-bonds -4 generate specific thioethylated monomers. After silylation (BSTFA), these monomers are analyzed by GPC-MS (electronic impact). The chromatograms reconstructed on the benzyl ions specific to each of them make it possible to evaluate unequivocally and without interference from the other constituents the relative frequency of the units having generated these monomers.

Figure 3: GPC-MS chromatographic profiles of control and under-expressing COMT lines: profiles reconstructed on the specific ions of each type of monomer.

Figure 4: HPLC analysis of products from the mild alkaline hydrolysis (1M NaOH, 2.5h, 40 C) samples of corn (stems + leaves) extracted.

P-coumaric acid E and Z: peaks 1 and 2. Ferulic acid E and Z: peaks 3 and 4.

Etalon (ethyl vanillin): peak 5. These profiles illustrate that the levels of PC (p-coumaric acid) decrease in the two ASCOMT1.2 and ASCOMT3.2 transgenic lines compared to the WT control.

Figure 5: Schematic representation of the plasmid constructs described in Example 1.

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Figure 6:
Figure 6A depicts a Northern blot analysis of the C-OMT 225 corn line at the 20 day stage.

 Figure 6B depicts a comparative Southern blot analysis between antisense line 225 and line 12, using four distinct restriction enzymes, respectively: AcoRV, KpnI, EcoRI and SacI.

Figure 7: Screening of a population of transgenic maize plants transformed respectively with the plasmids pProsper + pBar, prosperBar, pJim + pBar2 and pALIX + pBar2.

 The ordinate axis represents the COMT activity found in the maize plants analyzed, expressed in cpm / mg of protein.

 The histogram bars each represent a transgenic plant tested.

Figure 8: cribro-vascular bundles of adult leaves of control corn (12) and antisense C-OMT (225) stained according to the method of Maule. We see that the coloring is less intense in the sclerenchyma and in the xylem of 225; this observation suggests a decrease in S units in lignins of lineage 225.

DETAILED DESCRIPTION OF THE INVENTION
It has been shown according to the invention that, surprisingly, the induction of the modulation of the synthesis of an enzyme involved in the corn lignin biosynthesis pathway, the COMT methyltransferase, also called COMT, caused a modification. the ratio of S / G units in the composition of lignin and / or lignin content, compared to that found in wild maize, and that the change in S / G unit ratio and / or Lignin conferred on the maize plant and its processing products improved digestibility characteristics.

 By improved digestibility characteristics, a corn plant according to the invention is understood to mean that, with respect to a variety of wild maize plant, for example the aforementioned maize variety 12, the maize plant obtained

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 according to the invention has a digestibility index value dndf higher than the digestibility index value calculated from a corn plant of the wild variety.

 According to the invention, the value of the digestibility index dndf is calculated from ndf (neutral detergent fiber) whose method of production is described by Van Soest (1967) and dms (energetic solubility Aufrère) whose method of obtaining is described by Aufrère (1983), according to the formula dmdf = 100 * [dms- (100-ndf)] / ndf given by Struik (1985). The material used for the establishment of the ndf is a lyophilized powder of whole plants harvested at the flowering stage.

 Preferably, a corn plant obtained according to the invention has improved digestibility characteristics if the value of the index dndf is at least 80.

 Preferably, a corn plant according to the invention has a Klason lignin content, measured from the stems and leaves, which is less than 8.5, preferably less than 8, at 7.5, at 7.0 at 6.9 or 6.8, and preferably below 6.7.

 A preferred corn plant according to the invention has a Klason lignin content of between 6.1 and 6.6.

 To measure the Klason lignin content, those skilled in the art may advantageously refer to the technique described in Example 9.

 Surprisingly, it is shown for the first time according to the invention: a) that the inhibition of corn COMT methyltransferase enzyme made it possible to (i) reduce the ratio of units S / G units of lignin in this plant and (ii) reduce the lignin content in this plant; and b) that the reduction in the ratio of S units / units G of lignin and / or lignin content in maize resulted in maize plants with improved digestibility characteristics, compared to wild maize.

 Taking advantage of these unexpected results, the Applicant has developed a process for transforming maize plants in which the transformed maize plants have a ratio of S units / lignin G units and / or a modified lignin content, relative to to that found in wild corn plants.

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 Preferably, the ratio units S units G is less than the ratio units S / units G found in a wild maize plant.

 More specifically, it is shown according to the invention that transformed corn plants having improved digestibility characteristics can be obtained using an antisense polynucleotide.

 The subject of the invention is the use of a polynucleotide modulating the synthesis of corn comT methyltransferase encoded by the nucleotide sequence SEQ ID N'l, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between units S and G of the modified lignin and / or having a modified lignin content remaining compatible with the normal development of the plant.

Inhibition of COMT methyltransferase synthesis
According to a first embodiment, said polynucleotide inhibits the synthesis of corn COMT methyltransferase in the plant, when it is introduced by transformation into the cells of said plant.

 The antisense polynucleotide consists of a polynucleotide encoding an antisense RNA hybridizing with the messenger RNA constituting the transcription product of the corn COMT methyltransferase gene.

 The inhibition of the synthesis of the methyltransferase COMT can be obtained: (i) by transforming the corn cells with an antisense polynucleotide, as defined above, placed under the control of a functional regulatory sequence in the corn, said regulatory sequence may comprise a strong constitutive promoter and, where appropriate, also transcription activating sequences (functional enhancer sequences in corn), allowing a high level of expression of the number of copies of the mRNA of antisense methyltransferase COMT; - (ii) by stably introducing into the corn cells a plurality of copies of the antisense polynucleotide as defined above; - (iii) either by a combination of (i) and (ii), as described in the examples.

 Those skilled in the art can synthesize any antisense polynucleotide inhibiting the synthesis of COMT methyltransferase using its

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 general technical knowledge, from a nucleotide sequence encoding COMT, for example the sequence SEQ ID? 1.

 For the synthesis of a sense polynucleotide or an antisense polynucleotide specifically inhibiting the transcription or translation of a gene in a plant, those skilled in the art will advantageously refer to the review article by Matzke et al. (2001) as well as the content of articles referenced in this review article.

avantageusement aux articles de Baucher et al. (1999), de Zhong et al. (2000), de Pincon et al. (2001 a), de Blee et al. (2001), de Pincon et al. Still more specifically, for the construction or synthesis of a sense polynucleotide or antisense polynucleotide specifically inhibiting the transcription or translation of a gene encoding an enzyme involved in lignin biosynthesis in plants, including the COMT of certain plant species, the person skilled in the art can refer to

advantageously to articles Baucher et al. (1999), Zhong et al. (2000), from Pincon et al. (2001a), de Blee et al. (2001), de Pincon et al.

 (2001 b) or Lapierre et al. (1999).

 The antisense polynucleotide may also consist of the cDNA obtained from the mRNA constituting the transcription product of the gene encoding the methyltransferase COMT.

 It may also consist of a nucleotide fragment of this cDNA, preferably a nucleotide fragment of at least 100 consecutive bases of this cDNA, this length of the nucleotide fragment being sufficient to allow its specific hybridization with the corn COMT methyltransferase mRNA. and thus prevent its translation into protein.

 According to a first preferred aspect, the antisense polynucleotide comprises the nucleotide sequence SEQ ID. 2.

en le polynucléotide antisens de séquence SEQ ID ? 2. According to a second preferred aspect, the antisense polynucleotide consists

in the antisense polynucleotide of sequence SEQ ID? 2.

Increase (overproduction) of COMT methyltransferase
According to a second embodiment, said polynucleotide increases the synthesis of methyltransferase COMT in the plant, when it is introduced by transformation into the cells of said plant.

 According to this second embodiment, a polynucleotide encoding COMT maize methyltransferase is used, which causes production.

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 higher of this enzyme, when introduced by transformation into the plant.

 The synthesis or the isolation of a polynucleotide encoding corn COMT methyltransferase can be carried out by a person skilled in the art, using his general knowledge in molecular biology, notably thanks to his knowledge of the nucleotide sequence SEQ ID N l.

 The higher production of the COMT methyltransferase can be obtained: - (i) by transforming the corn cells with a polynucleotide encoding the COMT methyltransferase placed under the control of a functional regulatory sequence in maize, said control sequence being able to include a strong constitutive promoter and, where appropriate, also transcriptional activating sequences (functional enhancer sequences in corn), allowing a high level of expression of COMT methyltransferase; (ii) by stably introducing into the corn cells a plurality of copies of the polynucleotide encoding the COMT methyltransferase; - (iii) either by a combination of (i) and (ii).

 According to a first preferred aspect, the polynucleotide encoding the methyltransferase COMT comprises the nucleotide sequence SEQ ID N1.

 According to a second preferred aspect, the polynucleotide encoding the COMT methyltransferase consists of the nucleotide sequence SEQ ID N1.

METHODS FOR OBTAINING CORN PLANTS WITH ENHANCED DIGESTIBILITY CHARACTERISTICS.

 The invention also relates to a process for obtaining corn plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or whose lignin content is modified and compatible with the normal development of the plant, said method comprising a step of transforming a maize host cell with a polynucleotide selected from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding COMT maize methyltransferase ; b) a polynucleotide encoding corn COMT methyltransferase.

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According to a first embodiment of the above method may be characterized in that it comprises the following steps: a) transforming a maize host cell with an antisense polynucleotide specifically inhibiting the transcription or translation of the gene coding for the COMT methyltransferase; but. ; b) regenerating an entire plant from the recombinant host cell obtained in step a);
According to a first aspect, the modification of the lignin composition of the transformed corn plants that can be obtained by the above process consists in reducing the content of S units relative to the G units of the corn lignins, in order to obtain a lower S / G ratio than that of wild maize plants.

 Preferably, the S / G ratio is less than 40/60.

According to a second embodiment of the above method, said method is characterized in that it comprises the following steps: a) transforming a maize host cell with a polynucleotide encoding corn COMT methyltransferase; b) regenerating an entire plant from the recombinant host cell obtained in step a);
Preferably, the polynucleotide encoding corn COMT methyltransferase comprises the nucleotide sequence SEQ ID N'l.

 According to a particular embodiment, the process for obtaining maize plants transformed according to the invention is characterized in that it comprises the following additional step: c) selecting maize plants having integrated in their genome at least one copy of the antisense polynucleotide or at least one copy of the polynucleotide encoding corn COMT methyltransferase.

Expression cassettes comprising an antisense polynucleotide or a polynucleotide encoding corn COMT methyltransferase
Advantageously, the antisense polynucleotide or the polynucleotide encoding corn COMT methyltransferase is integrated into an expression cassette also comprising a nucleotide sequence regulating the expression of said polynucleotide in maize cells.

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 Advantageously, the antisense polynucleotide or the polynucleotide encoding the COMT methyltransferase is integrated in an expression cassette also comprising a nucleotide sequence with a function of transcription terminator.

 The expression cassette defined generally above may also be referred to as a DNA construct or DNA construct in the present description.

Agrobacterium tumefaciens. In yet another aspect, the antisense polynucleotide or the polynucleotide encoding the methyltransferase COMT, or the expression cassette in which is inserted respectively one or the other of these polynucleotides, is artificially inserted into a host cell.

Agrobacterium tumefaciens.

 The DNA constructs also include a gene promoter sequence and a terminator sequence operably linked to the DNA sequence to be transcribed. The gene promoter is generally located at the 5 'end to allow initiation of transcription of the DNA sequence. The promoter sequences are located in the non-coding regions of the genes but also sometimes in the introns (Luehrsen K, 1991) or in the coding regions, for example the PAL gene promoter of tomato (Bloksberg, 1991). When the construct includes an open reading phase in the sense orientation, it is necessary to use a full-length cDNA so that the protein can be translated. When the construct comprises an open-label antisense reading or a non-coding region, it is not essential to use a full-length cDNA, a partial sequence may suffice.

Promoters
Among the transcription promoters that may be used, mention may be made in particular of the so-called constitutive promoters, the so-called inductive promoters and the specific tissue promoters.

 A constitutive promoter allows strong expression of the transcript in all tissues of the regenerated plant and will be active in most environmental conditions, and in all stages of cellular transformations and differentiations. For example, the main dual constitutive promoters are:

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dans le plasmide pAct1-F4 décrit par Mc Elroy et al. (1991) - le promoteur ubiquitine 1 de maïs (Christensen et al., 1996) et d'autres régions initiatrices de la transcription de gènes de plantes variables connues de l'homme du métier. the 35S promoter, or advantageously the 35S double constitutive promoter (pd35S) of CaMV, described in the article by Kay et al., (1987) - the rice actin promoter followed by the rice actin intron content

in the plasmid pAct1-F4 described by Mc Elroy et al. (1991) - corn ubiquitin promoter 1 (Christensen et al., 1996) and other transcription promoter-variable gene regions known to those skilled in the art.

 Alternatively, it may be advantageous to use an inducible transcription promoter sequence capable of being controlled by the environmental conditions or the stage of development, for example the promoters phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG) , chitinases, glucanases, proteinase inhibitors (P1), genes of the PR1 family, opaline synthase (nos) or the vspB gene (US-5,770,349), all these promoters being recalled with the references corresponding publications in Table 3 of US Pat. No. 5,670. 349.

 Another category of promoters that can advantageously be used to carry out the method which is the subject of the invention groups the specific tissue promoters, in particular it can be promoters of the genes of the lignin biosynthesis pathway or, for example, the promoter of the promoter. alcohol dehydrogenase that expresses itself specifically in vascular tissues.

 Seed-specific promoters can also be mentioned (Datla, R. et al., 1997), in particular the promoters of napin (EP-0,255,378), glutenin and heliantinin (WO-92/17580). albumin (WO-98/45460), oleosin (WO-98/45461), ATS1 or ATS3 (WO-99/20775).

Terminating sequences
The terminator sequence used in the constructs that are the subject of the invention is located at the 3 'end of the sequence to be transcribed. It can come from the same gene as the promoter sequence or from a different gene.

 Among the transcription terminators that may be used include: the polyA 35S terminator of cauliflower mosaic virus (CaMV) described in the article by Franck et al., (1980), or

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 the polyA NOS terminator, which corresponds to the 3noncoding region of the opaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens opaline strain.

Selection marker
The constructs also include a selection marker for selecting plant cells transformed by these constructs. These selection markers are well known to those skilled in the art, particularly they confer resistance to one or more toxins, for example resistance to the herbicide BASTA (D. Moreover, the transformation of plant cells by the constructs, object of the invention can be controlled by other techniques well known in the art such as Southern and Western Blots.

Manufacture of an expression cassette
Techniques for operably linking all components of these constructs are well known to those skilled in the art and include the use of synthetic cloning sites comprising one or more endonuclease restriction sites as described, for example, by Maniatis et al. . (1989). The DNA constructs of the present invention may be linked to a vector having at least one replication system, for example with E. coli, after each manipulation, it is possible to clone and sequence the obtained construct to verify the accuracy of handling.

 The invention relates to the constructs as described above, and comprising at least one sequence chosen from the sequences SEQ ID No. 1 or 2 operatively linked upstream to a promoter and downstream to a terminator.

l'ADNc de la méthyltransférase (COMT ou AldOMT) et le terminateur de la Nopaline synthase, comme cela est montré dans l'exemple 1. Most preferably, the expression cassette comprises the promoter of the alcohol dehydrogenase specific to the vascular system,

the methyltransferase cDNA (COMT or AldOMT) and the Nopaline synthase terminator, as shown in Example 1.

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Expression vectors
The expression cassettes or DNA constructs as defined above may be included in expression vectors that can be used to transform an Agrobacterium tumefaciens cell or a maize host cell.

 The preferred bacterial vectors according to the invention are, for example, the pBR322 vectors (ATCC No. 37 017) or the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WI, USA). .

 Other commercialized vectors may also be mentioned, such as the vectors pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratagene).

 It may also be vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the Sf9 line (ATCC N CRL 1711) derived from Spodoptera frugiperda.

Preferentially, vectors specially adapted for expression of sequence of interest in plant cells, such as the following vectors: vector pBIN19 (BEVAN et al), marketed by CLONTECH (Palo Alto) will be used; , California, USA); vector pB1 101 (JEFFERSON, 1987), marketed by CLONTECH; vector pBI121 (JEFFERSON, 1987) marketed by the Company
CLONTECH; pEGFP vector; Yang et al. (1996), marketed by the Company
CLONTECH; pCAMBIA 1302 vector (HAJDUKI IEWICZ et al., 1994) - Intermediate and superbinary vectors derived from the pSB12 and pSB1 vectors described by Japan Tobacco (EP-0.672.752 and Ishida et al., 1996).

A very preferred first vector is the plasmid pAlix shown in FIG. 5, in which the sequence antisense polynucleotide has been inserted.
SEQ ID D? 2 inhibiting the synthesis of COMT maize methyltransferase ..

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 A second most preferred vector is the plasmid prosper represented in FIG. 5, in which the polynucleotide of sequence SEQ ID No. 1 coding the COMT corn methyltransferase was inserted.

Processing of maize plants
An increase in the synthesis of S and / or G units of lignin and / or lignin content in the plant can be obtained by integrating into the open reading phase a DNA construct in the sense orientation, in particular the sequence SEQ ID n'l. Transformation of a target plant with such a construct results in an increase in the number of copies of the transcript and thus an increase in the amount of enzyme. For example, an increase in S-unit synthesis can be achieved by integrating into the genome of the target plant the sense sequence of the methyltransferase encoded by SEQ ID No. 1.

 A reduction in the synthesis of S and / or G units of the lignin and / or the lignin content in the plant can be obtained by introducing into the open reading phase a DNA construct in antisense orientation, in particular any or part of the sequence SEQ ID No. 2. The transcribed RNA is complementary to the endogenous sequence of mRNA which will have the effect of reducing the number of copies of the transcript and thus of decreasing the amount of enzyme. For example, a decrease in S-unit synthesis can be achieved by integrating in the genome of the target plant the antisense sequence of the COMT methyltransferase encoded by SEQ ID No. 2. The DNA constructs can also include a nucleotide sequence. comprising a non-coding region of the COMT methyltransferase gene. Examples of non-coding regions that may be used in such constructs include introns and 5'or 3'non-coding sequences. Transformation of target plants with such DNA constructs can result in a reduction in the synthesis of lignin S and / or G units and / or lignin content by the co-deletion process, as have for example, Napoli et al. (1990) or Carvalho Niebel et al. (1995).

 Regulation of lignin S and / or G unit synthesis and / or lignin content can also be achieved by inserting said sequences into

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Utilisation d'un second polynucléotide modulant la synthèse d'une enzyme impliquée dans la biosynthèse de la lignine, autre que la méthyltransférase COMT
Selon encore un autre aspect du procédé d'obtention de plants de maïs possédant des caractéristiques de digestibilité améliorées, tel que défini ci-dessus, l'effet du polynucléotide antisens ou du polynucléotide codant la méthyltransférase COMT du maïs peut être complété par l'insertion dans le génome de la même plante de maïs d'un second polynucléotide dérivé du gène codant une enzyme impliquée dans la voie de biosynthèse de la lignine, autre que la méthyltransférase COMT. all or part (e.g., DNA or RNA) in ribozyme constructs (Haseloff et al., 1988).

Use of a second polynucleotide modulating the synthesis of an enzyme involved in the biosynthesis of lignin, other than COMT methyltransferase
According to yet another aspect of the process for obtaining maize plants having improved digestibility characteristics, as defined above, the effect of the antisense polynucleotide or the polynucleotide encoding corn COMT methyltransferase may be completed by the insertion. in the genome of the same corn plant a second polynucleotide derived from the gene encoding an enzyme involved in the lignin biosynthetic pathway, other than the COMT methyltransferase.

 Thus, the above method can also be characterized in that in step a), the maize host cell is also transformed with at least one second polynucleotide, specifically modulating the expression of an enzyme involved in the pathway. lignin biosynthesis, other than COMT maize methyltransferase.

 According to this particular aspect, said method is characterized in that the second polynucleotide is chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding the enzyme involved in the lignin biosynthetic pathway, other than the COMT methyltransferase corn; b) a polynucleotide encoding the enzyme involved in the lignin biosynthetic pathway, other than corn COMT methyltransferase.

nO d'accès X98083 et (c) l'enzyme F5H de Arabidopsis thaliana dont la séquence est référencée dans la base de données GenBank sous le nO d'accès U38416. The enzyme involved in the lignin biosynthetic pathway, other than corn COMT methyltransferase, is selected from: (a) the corn CAD enzyme whose sequence is referenced in the GenBank database under accession No. Y13733 (b) the maize CCR1 enzyme whose sequence is referenced in the GenBank database under the

Access No. X98083 and (c) the Arabidopsis thaliana F5H enzyme whose sequence is referenced in the GenBank database under access No. U38416.

 The enzyme involved in the lignin biosynthetic pathway, other than corn COMT methyltransferase, may also be selected from (d)

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 the maize CCoAOMT1 enzyme whose sequence is referenced in the GenBank database under access number AJ242980 and (e) the corn CCoAOMT2 enzyme whose sequence is referenced in the GenBank database under the nO Access AJ242981.

 Once obtained, the constructs are used to transform the plant cells. Advantageously, the transformation of the plant cells can be carried out by transfer of the abovementioned vectors into the plant protoplasts, in particular after incubation of the latter in a solution of polyethylene glycol in the presence of divalent cations (Ca 2+) (Schocher et al., 1986).

 The transformation of the plant cells can advantageously be carried out by electroporation, in particular according to the method described in the article by Fromm et al. (1985).

 The transformation of the plant cells can also be carried out using a particle gun allowing the projection, at very high speed, of metal particles coated with the DNA sequences of interest, thus delivering the sequences inside the nucleus. cell (Fromm et al., 1990, Finer et al., 1992).

 Another method of transformation of plant cells is that of cytoplasmic or nuclear microinjection (Neuhaus et al., 1987).

l'article d'An et al. (1986), ou encore Agrobacterium rhizogenes, notamment selon la méthode décrite dans l'article de Guerche et al. (1987). One of the methods of transforming plant cells that can be used in the context of the invention is the infection of plant cells by a bacterial cell host comprising the vector containing the sequence of interest. Advantageously, the abovementioned cellular host used is Agrobacterium tumefaciens, in particular according to the method described in

the article by An et al. (1986), or Agrobacterium rhizogenes, in particular according to the method described in the article by Guerche et al. (1987).

 Preferentially, the transformation of the plant cells is carried out by the transfer of the T region of the Agrobacterium tumefaciens Ti tumor-inducing extrachromosomal circular plasmid, using a binary system (Watson et al., 1994). To do this, two vectors are built. In one of these vectors, the T-DNA region was deleted except for the right and left edges, with a marker gene inserted between them to allow selection in plant cells. The other partner of the binary system is a Ti plasmid

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 helper, modified plasmid that no longer has T-DNA but still contains the virulence genes vir required for the transformation of the plant cell.

This plasmid is maintained in Agrobacterium.

 According to a preferred mode, the method described by Ishida et al. (1996) can be applied for the transformation of monocotyledons, especially maize.

 According to another protocol, the transformation is carried out according to the method described by Finer et al. (1992) using tungsten or gold particle gun.

 According to yet another aspect, the process for obtaining maize plants transformed according to the invention is characterized in that it comprises the following additional steps: d) crossing between them of two transformed plants as obtained at step b) or in step c) with a second maize plant; e) selection of plants homozygous for the transgene or transgenes.

 According to yet another aspect, the process for obtaining maize plants transformed according to the invention is characterized in that it comprises the following additional steps: f) crossing of a transformed maize plant obtained in step c) with a second maize plant; g) selecting maize plants from the cross of step f) and having retained the transgene.

choisie parmi les séquences SEQ ID n01 ou SEQ ID n 2. One of the embodiments of the method which is the subject of the invention concerns obtaining transgenic maize plants from transformed plant cells with a construction comprising at least one sequence

chosen from the sequences SEQ ID No. 01 or SEQ ID No. 2.

Host cells transformed with an antisense polynucleotide or a polynucleotide encoding COMT methyltransferase and transformed corn plants obtained from the transformed host cells.

 The subject of the invention is also a maize host cell or an Agrobacterium tumefaciens cell transformed with:

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 a) an antisense polynucleotide inhibiting the synthesis of COMT methyltransferase in a corn cell; b) a polynucleotide encoding the COMT methyltransferase, said polynucleotide (a) or said polynucleotide (b) being preferably placed under the control of a functional regulatory sequence in a corn cell, for example in an expression cassette or in a an expression vector as defined above.

 The present invention relates to transformed plant cells thus obtained. In particular, the corn cells having stably integrated in their genome constructions comprising all or part of the sequences SEQ ID n01 or n 2.

 The cells transformed by the constructs that are the subject of the present invention are selected by means of the selection marker, for example the resistance to BASTA®. The transgenic cells are then grown on a suitable medium to regenerate whole plants by methods well known to those skilled in the art. In the case of protoplasts, the cell wall can reform under the effect of appropriate osmotic conditions. In the case of seeds or embryos, a favorable medium for germination or callus initiation is employed. For explants, the medium used must allow regeneration.

 The principle of in vitro regeneration of maize plant cells is well known to those skilled in the art. The plants are regenerated from callus obtained after culture of anther cells, pollen grains, embryos or protoplasts. The cells are cultured on nutrient agar media adapted to each cell type under sterile conditions in petri dishes or culture tubes in a climatic chamber under favorable environmental conditions (adapted temperature and brightness). After transplanting, vitroplants are transplanted into a greenhouse.

 In particular, according to one aspect of the invention, maize plants with better digestibility and the content of S units and / or G is modified, including having a lower GIS ratio than plants whose ratio is not not modified can be obtained after hybridization with an individual whose COMT methyltransferase activity is modified by the principles of varietal selection well known to those skilled in the art.

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 Those skilled in the art may also employ vegetative propagation methods from corn plants whose COMT methyltransferase activity is modified, particularly to modify the ratio in S units / G units and / or lignin content. These methods may advantageously include certain steps in vitro. These may be cell regeneration steps with modified COMT methyltransferase activity. These may be methods of tissue culture, micro-propagation, meristem culture, embryo culture, corn plants with modified COMT methyltransferase activity. These may be methods of regenerating protoplasts from corn plants with modified COMT methyltransferase activity or from fusion with protoplasts from corn plants with modified COMT methyltransferase activity.

 The invention also relates to a transformed corn plant, obtainable by the method as defined above, which is characterized by improved digestibility characteristics.

 According to a first feature that can define it, a maize plant transformed according to the invention has improved digestibility characteristics and a dndf index value of at least 80.

 According to a second characteristic that can define it, a corn plant according to the invention has a Klason lignin content, measured from the stems and leaves, which is less than 8.5, preferably less than 8, at 7.5. at, 7.0, 6.9 or 6.8, and preferably less than 6.7.

 A preferred corn plant according to the invention has a Klason lignin content of between 6.1 and 6.6.

 According to a third characteristic that can define it, a corn plant transformed according to the invention has a S / G ratio of lignin of less than 40/60.

 The invention also relates to a portion of a transformed corn plant as defined above, for example a root, a seed, a leaf, a stem or a flower.

 It also relates to isolated corn cells obtained from a transformed corn plant as defined above.

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 The invention also relates to a recombinant expression vector comprising a polynucleotide selected from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding corn COMT methyltransferase; b) a polynucleotide encoding COMT methyltransferase; said polynucleotide being under the control of a functional regulatory sequence in maize.

 The subject of the present invention is also a DNA construct comprising a sense polynucleotide or an antisense polynucleotide specifically inhibiting the transcription or translation of the COMT gene of sequence SEQ ID No. 1, under the control of a functional promoter in maize. .

 It also relates to a DNA construct comprising a polynucleotide encoding COMT methyltransferase, under the control of a functional promoter in corn to increase the expression of said enzyme.

 The invention also relates to a transformation product obtained from a transformed maize plant or a part of a transformed maize plant, as defined above. Such a transformation product is characterized in particular in that the lignin it contains has a modified ratio between the units S and the units G.

 Preferably, the S / G ratio is less than 40/60.

 The said transformation product includes feed for livestock, obtained from maize plants whose contents in S units or G units are modified and / or the lignin content of which is modified.

 The subject of the invention is also lignin purified from a transformed maize plant or from a maize plant part as defined above, characterized in that it notably has a ratio between the S units and G units that is modified, compared to the S / G ratio found in the lignin of wild corn plants.

 Preferably, the purified lignin above is characterized in that the S / G ratio is less than 40/60.

 The present invention is illustrated, without being limited, to the following examples.

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EXAMPLES Example 1 Construction of transformation vectors: Several vectors were constructed in order to obtain the transformation vector.

TOM22 (5'-CTGCTGGAGGTGCTGCAGAAG-3'-SEQ ID N'3) et TOM23 (5'-CTCCTTGCCCCCGGGGTTGTG-3-SEQ ID N 4'), permettant d'introduire respectivement les sites Pstl et Smal, en 5'et en 3'd'un fragment de 0.85 Kpb compris entre les positions 0.17 et 1.04 Kpb de l'ADNc. Ce fragment est ensuite sous-cloné dans le vecteur pGemT (Promega), et le plasmide obtenu est digéré par Pstl et Smal. Le fragment libéré est sous cloné dans pUC18-Nos aux sites Pstl et Smal, générant ainsi le plasmide pTMO-Nos. - Plasmid pProsper [Figure 5J
A PstI-HindIII fragment of 0.26 Kpb corresponding to the terminator of the opaline synthase gene (T-Nos) is subcloned in pUC18 (Yannisch-Perron C. et al., 1985) between the PstI and HindIII sites, thus giving the vector pUC18-Nos. Plasmid pMC1 (Collazo P. et al., 1992), corresponding to the cDNA of C-OMT (1.4 kb) inserted at the pstI site of pUC18. PCR amplification is carried out on this plasmid with the oligonucleotides

TOM22 (5'-CTGCTGGAGGTGCTGCAGAAG-3'-SEQ ID N'3) and TOM23 (5'-CTCCTTGCCCCCGGGGTTGTG-3-SEQ ID N 4 '), making it possible to introduce respectively the PstI and SmaI sites, in 5' and in 3 of a 0.85 Kpb fragment between the 0.17 and 1.04 Kpb positions of the cDNA. This fragment is then subcloned into the pGemT vector (Promega), and the resulting plasmid is digested with PstI and SmaI. The released fragment is subcloned into pUC18-Nos at the PstI and SmaI sites, thereby generating plasmid pTMO-Nos.

 Plasmid pBARGUS (Fromm et al., 1990) contains a CaMV35S-intron Adhl-BAR-Tnos promoter cassette as well as an Adhl-Intron Adhl-GUS-Tnos promoter cassette. The latter is released by an EcoRI and HindIII digestion. In a second step, a partial EcoRI / Pvull digestion of the same fragment makes it possible to release a 1.75 kbp fragment corresponding to the Adhl promoter coupled to the Adhl intron. This fragment is then subcloned into pTMO-Nos between EcoRI and Smal. The plasmid obtained is prosper.

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- Plasmid pAlix [Figure 5 /
Plasmid PMC1 is digested with HindIII) and XbaI to release the full length cDNA (1.4 Kbp) of C-OMT. The plasmid pActin-BAR (Wu, Cornell University, New York) consists of the plasmid psP72 containing an actin-actin-actin-BAR-Tnos promoter cassette This plasmid is digested with HindIII and XbaI to remove the BAR fragment, and the fragment HindIII-XbaI of 1.4 Kbp of C-OMT is subcloned between these two sites, thus giving the plasmid pAlix.

-Plasmid pBar2 [Figure 5]
The plasmid pBARGUS is digested with HindIII, to release the CaMV35S -Intron Adh1-BAR-Tnos promoter cassette of 2.13 Kpb. The thus released cassette is subcloned into pUC18 at the HindIII site to give plasmid pBar2.

PBar Plasmid [Figure 5]
The construction of plasmid pBar is described by Peter Eckes (Biology Research Center H 872 N, Hoechst AG 6230 Frankfurt 80 / EP-0.275, 957).

Example 2: Transformation by Aarobacterium tumefaciens
The transformation technique described by Ishida et al. (1996) is used from immature embryos 10 days after fertilization.

 All media used are referenced in the reference cited.

The transformation begins with a c-culture phase where the immature embryos of the maize plants are brought into contact for at least 5 minutes with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors. The super-plasmid is the result of homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest and / or the selection marker derived from the plasmids described in the preceding examples, and the pSB1 vector of
Japan Tobacco (EP 672 752) which contains: the virB and virG genes of plasmid pTiBo542 present in the supervirulent strain A281

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 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination.

 The embryos are then placed on LSAs medium for 3 days in the dark and at 25 C. A first selection is performed on the transformed calli: the embryogenic calli are transferred to LSD5 medium containing phosphinotricin 5 mg! 1 and cefotaxime 250 mg! 1 (elimination or limitation of contamination with Agrobacterium tumefaciens). This step is carried out for 2 weeks in the dark and at 25 ° C. The second selection step is carried out by transfer of the embryos which developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as before. The third step of selection consists in excising the callus type 1 (fragments of 1 to 2 mm) and transfer them 3 weeks in the dark and 250C on LSD 10 medium in the presence of cefotaxime.

 Regeneration of the seedlings is performed by excising the proliferating type 1 calli and transferring them to LSZ medium in the presence of 5 mg / l phosphinotricin and cefotaxime for 2 weeks at 220C under continuous light.

 The regenerated seedlings are transferred to RM + G2 medium containing 100 mg! 1 Augmentin for 2 weeks at 220C and under continuous illumination for the development stage. The resulting plants are then transferred to the phytotron for acclimation.

Example 3: Bombardment Transformation
Callus genotype Iz, aged 2 months, obtained after immature embryos were cultured on a callogenesis medium are used for the transformation.

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Plasmolysing treatment reduces the volume of the vacuole and thus limits cell burst during firing (4-hour pretreatment and a 16-hour post-treatment on 0.2M Sorbitol and 0.2M Mannitol).

The plant material is homogeneously distributed over all of the boxes.

15 mg of sterile tungsten microbeads are mixed in 150 liters of sterile milliQ water.

- 2, 5 ul d'ADN du plasmide portant le gène de sélection (à 1 pg/pl), - de CaCI2 à 2, 5M, -10 pI de spermidine à 0,1 M, Ce mélange est laissé à décanter dans la glace pendant au moins 5 minutes. For 5 shots, the following mixture is prepared: 25 μl of tungsten microbeads, 2.5 μl of plasmid DNA carrying the gene of interest (at 1 μg / μl),

2.5 μl of plasmid DNA carrying the selection gene (at 1 μg / μl), CaCl 2 at 2.5 M, 10 μl of 0.1 M spermidine, this mixture is allowed to decant in the ice for at least 5 minutes.

Five Petri dishes containing the material to be bombarded are placed on agar plates at 15g / L.

 50ul of supernatant from the decanted tube are removed and the remaining mixture is well vortexed, then 2 1-11 of it is deposited on the grid of the sterile syringe, which is screwed onto the support in the barrel.

A sterile gauze filter is placed on the petri dish which is placed 19 cm into the barrel enclosure. The vacuum is pushed inside the enclosure at the pressure of 25 mbar The firing is triggered. The pressure of the helium in the barrel is 8 bars.

The bombarded plant material is returned to its original petri dish (Mannitol / Sorbitol). The dishes are placed at 26 C and in the dark for 16 hours for post-bombardment plasmolysing treatment (mannitol / sorbitol).

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The selection pressure is applied 16 hours after firing and is maintained at the same concentration of the selective agent (bialaphos at 5mg / L) during all regeneration steps. The seedlings are then transplanted into potting soil (small pots) for 1 week and then transferred in 20-liter pots (peat-pozzolan mixture) in the transgenic greenhouse or in an air-conditioned room (Temperature 24 C day / 20 C night, photoperiod 16h day / 8h night, hygrometry 80%). The plants are self-fertilized or crossed.

Example 4: Analysis of Northern Biot RNAs and Southern Blot Genomic DNA A. Materials and Methods
The RNAs are isolated by the Extract-all solution (Eurobio), according to the protocol of the supplier. Additional precipitation with 0.2 M NaCl with 2 volumes of absolute ethanol is carried out. 1 μg of RNA are separated on formaldehyde gels (Sambrook et al., 1989). Total genomic DNA is extracted from 1 g of leaves by the method described in Dellaporta et al. (1983). After a phenol chloroform purification step, 10 μg of DNA are digested with 40 units of restriction enzyme (Promega), and separated on TAE agarose gel 1% (w / v). RNAs or DNAs are transferred to nylon membranes (Hybond-N +, Amersham), and hybridized with 32p-dCTP-labeled DNA probes by the Promega premium-a-gene labeling system kit. The hybridization signal is determined by contacting the membranes with Biomax films.
Kodak MS in an autoradiography cassette.

B. Results
Figure 6A depicts a Northern blot analysis of the C-OMT 225 corn line at the 20 day stage.

 RNA (R), collar (c), and wound (H) RNAs of line 225 and control line I2 were deposited on agarose electrophoresis gel.

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After migration and incubation for 10 minutes in a BET solution at 0.1 μg / ml, the gel was photographed under UV in order to estimate the relative amounts of ribosomal RNAs (rRNA). The hybridization of the membrane after transfer with a 32p-labeled cDNA probe makes it possible to observe a sharp decrease in C-OMT transcripts in all in all the organs tested (R, C, H) in line 225, compared to 12 .

 Figure 6B depicts a comparative Southern blot analysis between the antisense line 225 and the Is line, using four distinct restriction enzymes, respectively: EcoRV, KpnI, EcoRI and SacI.

 The probe used is a 1.2 Kb fragment located in the 3 'region of the maize C-OMT cDNA.

 The bands revealed on line 12 are due to the endogenous COMT gene.

 The supernumerary bands present for line 225 correspond to the transgenic sequences: the enzymes EcoRV, KpnI and SacI do not cut in the transgene. An Eco RI site is present in the 5 'part of transgenic construction. Depending on the case, 3 bands (EcoRI, EcoRV) or 2 bands (Kpnl, Sacl) supernumerary appear for the line 225.

 The results show that line 225 was transformed with 2 or 3 copies of the transgene.

Example 5 Enzymatic Analysis of COMT A. Materials and Methods
The protocol used is derived from Atanassova et al. (1995). Briefly, young plants of 20 days (post-sowing) are harvested by cutting at the collar, then the leaves are removed from the aerial part (cut at the level of the ligule). The remaining fragment, about 15 cm long is fixed in liquid nitrogen and ground in a mortar with a pestle. The powder obtained is homogenized in the extraction buffer Na2HPO, JNaH2PO4 0. 1M pH7.5, PVPP 5% (m / v), DTT (10 mM) at a rate of
1 mL of buffer for 300 mg of powder. After 1/2 hour of incubation at 4 C, two successive centrifugations (3000 g / 10 min / 4 C) are performed to remove the insoluble material. 40 μl of crude extract thus obtained is added to

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960 uL de mélange réactionnel (Acide caféique 3mM, 3H-SAM 0. 1 uCi, SAM 50uM, DTT 10mM, Tampon Na2HP041 NaH2P04 0. 1M pH7. 5 qsp 960jjL) et l'ensemble est incubé 1 h à 37 C. La réaction est arrêtée avec 100uL d'acide sulfurique 9N et 5 mL de scintillant organique (OCS, Amersham) sont ajoutés au mélange. L'ensemble est homogénéisé pour faire passer l'acide férulique tritié, produit de la réaction, dans le scintillant organique. La quantification de la radioactivité de l'acide férulique est réalisée avec un compteur à scintillation Beckman. Les protéines sont dosées selon la méthode de Bradford (1976) avec le réactif de Biorad.

960 μl of reaction mixture (3 mM caffeic acid, 3 H-SAM 0.1 μCi, 50 μM SAM, 10 mM DTT, Na 2 HPO 4 NaH 2 PO 4 0. 1M pH7 buffer, qsp 960 μL) and the whole is incubated for 1 h at 37 ° C. The reaction is stopped with 100 μl of 9N sulfuric acid and 5 ml of organic scintillant (OCS, Amersham) are added to the mixture. The mixture is homogenized to pass the tritiated ferulic acid, product of the reaction, in the organic scintillant. Quantification of the radioactivity of ferulic acid is performed with a Beckman scintillation counter. The proteins are assayed according to the method of Bradford (1976) with the Biorad reagent.

B. Results Screening of transgenic plants on the basis of C-OMT activity (Figure 7) 1-Selection of the part of the plant used for the measurement of COMT activity Plants were harvested at a young stage (aged 20 days after sowing), which implies a short delay between sowing and analysis, and which makes it possible to cultivate a large number of plants simultaneously. The stem and the young rolled leaves were used as a mixture for this work. Ligulated leaves were removed.

2-C-OMT activity in control plants used for screening
The line used as a control in the screen is 12. The histogram bar of the controls 12 results from the average of the results obtained on 12 plants analyzed twice. For these controls, the activity values varied from 4385 to 7225 cpm / mg of protein, the average being 5998 cpm / mg with a standard deviation of 1039. This first result makes it possible to estimate the variability of the C-OMT activity between different plants harvested at the same time. We see that it is relatively small.

The mutant corn line bm3 was used for comparison. It was interesting to validate the protocol for measuring C-OMT activity, used for the first time on maize, using the mutant bm3, which shows an activity
C-OMT void. Three bm3 plants were analyzed, and a level

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 very low activity corresponding to background noise. The crude protein extracts of the bm3 plants are therefore excellent negative witnesses in this work.

3-activity C-OMT in transgenic populations
Results relating to the analysis of 32 different transformation events are summarized in Figure 7. Since the analysis was made in some cases on two batches of seeds of the same transformation event.

Three plants from the same lot were analyzed so that a standard deviation could be established. Each crude extract was used for two activity determinations.

A total of 20 events with the promoter ADH-ASOMT (prosper and pProsper-Bar) were analyzed. A significant reduction in COMT activity, of the order of 70%, has been achieved for some events.

Example 6: Histoloacic Analysis A. Materials and Methods • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 15N for 3 minutes, rinsed in distilled water, and incubated in 5% (w / v) NaHCO 3 solution.

Phloroglucinol stain: The sections are immersed in a solution of phloroglucinol in hydrochloric solution (Prolabo).

The photographs are made with a camera.

B. Results
The results are shown in FIG. 8, which shows photon microscopy images of the cribro-vascular bundles of adult maize leaves, respectively of the control maize line Is and the maize line 225, which has been transformed with a polynucleotide. antigen inhibiting COMT methyltransferase.

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 In Figure 8, it is seen that the staining is less intense in the sclerenchyma and in the xylem of the 225 line than in the sclerenchyma and xylem of the control line 12.

 This reflects a decrease in S units in the lignins of the transgenic maize 225 variety.

Example 7: Test of digestibility of plants by evaluation of the enzymatic solubility A. Materials and Methods
This method makes it possible to estimate a potential digestibility by the activity of cellulolytic enzymes on the plant wall. The samples are placed in sealed sachets and immersed in the enzyme solutions. The reactions take place in a DAIZY incubator which can contain four 2-liter flasks, rotated, kept at 40 ° C. and can contain from 20 to 80 sachets. The freeze-dried powder samples are incubated overnight at 70 ° C. in a crystallizer. Then, 0.5 g of powder (initial mass) are deposited in a bag (F57, Ankom) which is closed by welding, incubated for 24 hours at 70 ° C. and then weighed after cooling (P1). The sachet is then incubated in a solution of pepsin (Merck) at 20 g / l of 0.1 N HCl at 40 ° C. for 24 hours and then at 80 ° C. for 14 hours. The whole is then rinsed with water at 35 C and wrung. This step is repeated once. The sachet is then incubated in a solution of cellulase (Onozuka R10 Yakult, Biochemical Co, Ltd.) at 1 g / L with amyloglucosidase (Merck).
0. 1 g / L in sodium acetate / acetic acid buffer (2.95 mL of 96% acetic acid and 6.8 g of sodium acetate in 1 L of demineralised water), at 40 C for 24 h, then rinsed and wrung twice, before being dried in an oven at 70 C for 24 hours. The bag is weighed (P2). The solubility (in% of the dry mass) is equal to (P1-P2) / (initial mass × 100).

B. Results
Comparative results of digestibility are presented in Table
1 below.

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Table 1: Digestibility index of maize plants

<Tb>
<tb> Line <SEP> Neutral <SEP> detergent <SEP> fiber <SEP> Digestibility <SEP> (dndf)
<tb> (ndf)
<tb> 225 <SEP> 1 <SEP> 47. <SEP> 69 <SEP> 86. <SEP> 24
<tb> 225 <SEP> (2) <SEP> 43. <SEP> 65 <SEP> 82. <SEP> 20
<tb> control <SEP> 1246. <SEP> 8577. <SEP> 61
<Tb>

In Table 1, the index ndf represents the percentage of nondigestible fraction measured from lyophilized powder of whole plants harvested at the flowering stage.

 In Table 1, the index dndf represents the digestibility index calculated from the value of ndf (neutral detergent fiber) and dms (energetic solubility Aufrère) according to the formula of Struik (1985).

 The results of Table 1 show the higher digestibility of the two transgenic maize lines 225 (1) and 225 (2) which were transformed with an antisense construct according to the invention, compared to the control corn variety WT, the line of corn Iz.

Example 8: Evaluation of the content. composition and structure of transgenic corneal maize under-expressing COMT: evaluation by GPC-MS of monomeric products derived from their thioacidolysis 11 Liqnine study: content and structure Three maize lines, one control (WT) and two lines COMT antisense of the same transformation event with a residual activity of 40% (3.2 and 1.2) harvested at the flowering stage, freeze-dried and crushed were analyzed.

The samples were extracted with an acetone / water mixture (5/1) in order to eliminate most of the soluble compounds (pigments, lipids, etc.) by a relatively fast protocol. The results are presented in the
Table 2: The "residue" of the extraction represents roughly the percentage of walls in the sample.

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Table 2: Results of Extraction of Control (WT) and Transgenic Corn

<Tb>
<tb> WT <SEP> ASCOMT1. <SEP> 2 <SEP> ASCOMT3. <SEP> 2
<tb>% <SEP> walls <SEP> 57. <SEP> 7 <SEP> 52. <SEP> 5 <SEP> 55. <SEP> 2
Extractive <tb>% <SEP><SEP> 42.3 <SEP> 47. <SEP> 5 <SEP> 44. <SEP> 8
<Tb>
The relatively high extractable content is attributable to the presence of the leaves.

The observed values are quite similar for the 3 lines.

The Klason lignin assay was performed on the extracted samples. It is expressed in weight percent of these samples (Table 3).

Table 3: Klason lignin content of corn samples (stems + leaves) extracted. Results expressed in% by weight of the extracted walls

<Tb>
<tb> WT <SEP> ASCOMT1. <SEP> 2 <SEP> ASCOMT3. <SEP> 2
<tb> Assay <SEP> 1 <SEP> 8. <SEP> 89 <SEP> 6. <SEP> 11 <SEP> 6. <SEP> 50
<tb> Assay <SEP> 2 <SEP> 8. <SEP> 63 <SEP> 6. <SEP> 34 <SEP> 6. <SEP> 57
<tb> Average <SEP> (range-8. <SEP> 76 <SEP> (0.18) <SEP> 6.23 <SEP> (0.16) <SEP> 6.54 <SEP> (0.05)
<tb> type)
<Tb>
The lignin contents are lower than in the case of stems harvested at maturity: the extracted samples come indeed from (leaves + stems) harvested at the flowering stage. Both transgenic lines have a lower lignin content (25 to 30% less) than the control line.

As the extractable content is similar for the 3 lines (Table 2), the difference between WT and antisense is maintained when the calculation is performed on the non-extracted sample In order to evaluate the impact of the transformation on lignin structure whole plants (leaves + stems) were subjected to thioacidolysis (Lapierre et al., Plant Physiol119, 153-163, 1999), followed by GC-MS analysis of their trimethylsilyl derivatives (Saturn Varian, electronic impact, 45 -650 m / z). The

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 Relative frequency of the p-hydroxyphenyl H, guaiacyl G, S-syringyl and 5-hydroxyguanal 5-OH G units engaged only in -0-4 bonds was determined on the chromatograms reconstructed on the most specific ions of the derivatives they generate. (Figure 2). These ions, corresponding to the base peak of the mass spectrum, make it possible to unequivocally evaluate the proportions of the H / G / S / 5-OHG units bound in -0-4.

The results reported in Table 4 and Figure 3 unequivocally demonstrate the effectiveness of the transformation to under-express COMT activity. Transgenic lines 3.2 and 1.2 show a collapse of the frequency of S units (divided by 2) and especially the appearance in their lignins of OH-G units in a substantial amount (multiplied by a factor of between 10 and 20). The effect is a little more marked in the case of line 1.2. The differences observed between Tables 3 and 4 for the controls are attributable to the differences in the organs analyzed, the stage of harvest and the line.

Table 4. Structure of maize lignins (stems + leaves harvested at flowering) of control and under-expressing COMT lines: Relative frequency of H, G, S and 5-OHG units bound in -0-4

<Tb>
<tb> Sample <SEP> Witness <SEP> AS <SEP> COMT <SEP> 3. <SEP> 2 <SEP> AS <SEP> COMT <SEP> 1. <SEP> 2
<tb>% <SEP> units <SEP> 3, <SEP> 2 <SEP> 2.5 <SEP> 2,4
<tb>% <SEP> units <SEP> 56, <SEP> 0 <SEP> 68.7 <SEP> 70.1
<tb>% <SEP> Units <SEP> S <SEP> 40, <SEP> 5 <SEP> 24.4 <SEP> 20.4
<tb>% <SEP> Units <SEP> 50H <SEP> G <SEP> 0.3 <SEP> 4.4 <SEP> 7.1
<tb> SIG <SEP> 0.72 <SEP> 0.36 <SEP> 0.29
<Tb>

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21 Etude des esters p-OH cinnamiques liés aux parois de maïs extraites Les esters p-hydroxycinnamiques liés aux parois ont été libérés par hydrolyse alcaline (NaOH 1 M, 2. 5 h, 40 C, 20 mg d'échantillon extrait traité par 2 ml de soude, en présence de 0. 2 mg étalon interne éthylvanilline, avec agitation magnétique et sous N2). L'hydrolysat a été acidifié par HCI 2 M (1. 5 ml), ultrafiltré sur Millex H25 NS (Millipore). Les composés phénoliques ont été extraits par extraction en phase solide sur Sep-Pak C18 (Classic, Waters Millipore), selon un protocole adapté de Beveridge et al. (2000, Food Res. Internat., 33, 775-783). Le conditionnement de la cartouche Sep-pak a été réalisé par 2*5 ml MeOH, 2*5ml eau (milli), 2*5 mi tampon formiate pH 3, avant passage de l'hydrolysat acidifié, suivi d'un lavage par 3 ml tampon formiate. L'élution des composés phénoliques retenus intégralement sur la cartouche (nous l'avons vérifié) a été réalisée par 1 mi CH3CN. L'échantillon recueilli a été dilué v/v par le solvant de CLHP avant d'être ultrafiltré à nouveau et analysé par CLHP en phase inverse (20 ul injectés). Un traitement identique a été réalisé sur un mélange étalon témoin contenant des quantités équipondérales d'acide p-coumarique (PC), d'acide férulique (Fe) et d'éthylvanilline (EtV), pour réaliser la calibration. L'analyse CLHP a été réalisée sur colonne Lichrocart RP18 Merck (5 pm, 250 * 4 mm), utilisée en conditions isocratiques, avec élution par le mélange eau/CH3CN/CH3COOH (78/20/2, v/v) à un débit de 1. 3 ml/min et avec détection en sortie de colonne à 280 nm. Les résultats sont rapportés dans le tableau 5 et illustrés en Figure 4.

Study of p-OH cinnamic esters bound to the extracted corn walls The p-hydroxycinnamic esters bound to the walls were released by alkaline hydrolysis (1 M NaOH, 2.5 h, 40 ° C., 20 mg of 2-treated extract sample). ml of sodium hydroxide, in the presence of 0.2 mg internal standard ethylvanillin, with magnetic stirring and under N 2). The hydrolyzate was acidified with 2 M HCl (1.5 ml), ultrafiltered on Millex H25 NS (Millipore). The phenolic compounds were extracted by solid phase extraction on Sep-Pak C18 (Classic, Waters Millipore), according to a protocol adapted from Beveridge et al. (2000, Food Res Internat., 33, 775-783). The conditioning of the Sep-Pak cartridge was carried out with 2 * 5 ml MeOH, 2 * 5 ml water (milli), 2 * 5 mi formate buffer pH 3, before passing the acidified hydrolyzate, followed by washing with 3 ml formate buffer. The elution of the phenolic compounds retained integrally on the cartridge (we verified it) was carried out with 1 ml CH3CN. The collected sample was diluted v / v with the HPLC solvent before being ultrafiltered again and analyzed by reverse phase HPLC (20 μl injected). Identical treatment was carried out on a control standard mixture containing equal amounts of p-coumaric acid (PC), ferulic acid (Fe) and ethylvanillin (EtV) to perform the calibration. HPLC analysis was carried out on a Merck Lichrocart RP18 column (5 μm, 250 × 4 mm), used under isocratic conditions, eluting with water / CH 3 CN / CH 3 COOH (78/20/2, v / v) at room temperature. flow rate of 1.3 ml / min and with detection at the column outlet at 280 nm. The results are reported in Table 5 and illustrated in Figure 4.

Table 5: Quantity of p-coumaric acids (PC) and ferulic acids (FE) released by mild alkaline hydrolysis (1 M NaOH, 2.5 h, 40 C) of corn samples (stems + leaves) extracted. Results expressed in% by weight of the extracted walls

<Tb>
<tb> WT <SEP> ASCOMT1. <SEP> 2 <SEP> ASCOMT3. <SEP> 2
<tb> Try <SEP> PC <SEP> FE <SEP> PC <SEP> FE <SEP> PC <SEP> FE
<tb> Assay <SEP> 1 <SEP> 0.81 <SEP> 0.89 <SEP> 0.55 <SEP> 1.03 <SEP> 0.54 <SEP> 1.06
<tb> Assay <SEP> 2 <SEP> 0.80 <SEP> 0.85 <SEP> 0.56 <SEP> 1.05 <SEP> 0.54 <SEP> 1.04
<tb> Average <SEP> 0.80 <SEP> 0.86 <SEP> 0.56 <SEP> 1.04 <SEP> 0.54 <SEP> 1.05
<Tb>

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A nouveau, les résultats présentent une plus faible teneur en esters pcoumariques que les lignées témoins analogues, tandis que les teneurs en esters féruliques sont peu affectées par la déficience en activité COMT. Le résultat global est une diminution du rapport PC/FE.

Again, the results show a lower PCO content than similar control lines, whereas ferulic ester levels are little affected by the COMT activity deficiency. The overall result is a decrease in PC / FE ratio.

Ralph et al. have repeatedly shown that a part of the acyl acyl acid acyl units of lignins (by NMR and thioacidolysis): the decline in PC esters observed here goes together with the decline of units S.

Ferulic esters are associated with polysaccharides and can be linked to lignins by ether bonds (Jacquet et al., 1995). The lower content of transgenic corn in lignins limits this linkage and is likely to reflect the fact that more ferulic acid is released by alkaline hydrolysis of the transgenic lines compared to the control.

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Claims (31)

 1. Use of a polynucleotide modulating corn synthesis of methyltransferase COMT encoded by the nucleotide sequence SEQ ID NI, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between the units S and G lignin modified and / or having a modified lignin content remaining compatible with the normal development of the plant. 2. Use according to claim 1, characterized in that said polynucleotide is an antisense polynucleotide inhibiting the synthesis of COMT maize methyltransferase. 3. Use according to claim 2, characterized in that the

 antisense polynucleotide comprises the nucleotide sequence SEQ ID? 2. 4. Use according to claim 1, characterized in that said polynucleotide encodes corn COMT methyltransferase, and causes production. higher of this enzyme in the plant. 5. Use according to claim 4, characterized in that the polynucleotide encoding the methyltransferase COMT comprises the nucleotide sequence SEQ ID Nos. 6. Process for obtaining corn plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or whose lignin content is modified and compatible with the normal development of the plant, said method comprising a step of transforming a maize host cell with a polynucleotide selected from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding corn COMT methyltransferase; b) a polynucleotide encoding COMT maize methyltransferase. <Desc / Clms Page number 47> 7. Method according to claim 6, characterized in that it comprises the following steps: a) transforming a maize host cell with an antisense polynucleotide specifically inhibiting the translation of the gene coding for corn COMT methyltransferase. ; b) regenerating an entire plant from the recombinant host cell obtained in step a); 8. Method according to claim 7, characterized in that the antisense polynucleotide comprises the nucleotide sequence SEQ ID D? 2. 9. Method according to claim 6, characterized in that it comprises the following steps: a) transforming a maize host cell with a polynucleotide encoding corn COMT methyltransferase; b) regenerating an entire plant from the recombinant host cell obtained in step a); 10. Method according to claim 9, characterized in that the polynucleotide encoding corn comT methyltransferase comprises the nucleotide sequence SEQ ID N01.  11. Method according to one of claims 6 to 10, characterized in that it comprises the following additional step: c) selecting maize plants having integrated in their genome at least one copy of the antisense polynucleotide or at least one copy of the polynucleotide encoding COMT maize methyltransferase.  12. Method according to one of claims 6 to 11, characterized in that the antisense polynucleotide or the polynucleotide encoding the methyltransferase COMT corn is integrated into an expression cassette comprising a nucleotide sequence regulating the expression of said polynucleotide in corn cells. <Desc / Clms Page number 48> 13. Method according to one of claims 6 to 12, characterized in that the antisense polynucleotide or the polynucleotide encoding corn COMT methyltransferase is artificially inserted into an Agrobacterium cell.

 tumefaciens. 14. Method according to one of claims 6 to 13, characterized in that in step a), the corn host cell is also transformed with at least one second polynucleotide, specifically modulating the expression of an enzyme involved. in the lignin biosynthetic pathway, other than corn COMT methyltransferase. 15. Method according to claim 14, characterized in that the second polynucleotide is chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding for the enzyme involved in the lignin biosynthesis pathway, other than the COMT methyltransferase; corn; b) a polynucleotide encoding the enzyme involved in the lignin biosynthetic pathway, other than corn COMT methyltransferase. 16. The method of claim 15, characterized in that the enzyme involved in the lignin biosynthetic pathway encodes an enzyme other than corn COMT methyltransferase. 17. Method according to one of claims 6 rai 16, characterized in that it comprises the following additional steps: d) crossing between them of two transformed plants as obtained in step b) or step c ) with a second maize plant; e) selection of plants homozygous for the transgene or transgenes. 18. Process for obtaining a transformed plant according to one of claims 11 to 16, characterized in that it comprises the following additional steps: f) crossing of a transformed corn plant obtained in step c) with a second maize plant; g) selecting the corn plants resulting from the crossing of step f) and having retained the transgene. <Desc / Clms Page number 49> 19. Corn cells obtainable by the method according to one of claims 6 to 18. 20. Processed maize plant obtainable from the cells according to claim 19. 21. Part of a transformed corn plant according to claim 20, in particular, root, seed, stem, leaf or flower. 22. A DNA construct comprising a polynucleotide modulating the corn COMT methyltransferase synthesis, selected from the sequences

 nucleotides SEQ ID N01 or SEQ ID? Or a fragment thereof, under the control of regulatory sequences operably linked to the DNA sequence to be transcribed. 23. DNA construct according to claim 22, characterized in that it also comprises a second polynucleotide encoding an enzyme involved in the biosynthesis of lignins, other than corn COMT methyltransferase. 24. A recombinant expression vector comprising a polynucleotide selected from: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding for Corn COMT methyltransferase; b) a polynucleotide encoding methyltransferase COMT; said polynucleotide being under the control of a functional regulatory sequence in maize. 25. Recombinant expression vector according to claim 24, characterized in that it comprises a second polynucleotide, said polynucleotide being chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding for the enzyme involved in the lignin biosynthesis, other than COMT maize methyltransferase; <Desc / Clms Page number 50>  b) a polynucleotide encoding the enzyme involved in the lignin biosynthetic pathway, other than corn COMT methyltransferase. 26. A corn host cell or an Agrobacterium tumefaciens cell transformed with: a) an antisense polynucleotide inhibiting the synthesis of methyltransferase COMT in a corn cell; b) a polynucleotide encoding the COMT methyltransferase, said polynucleotide (a) or said polynucleotide (b) being preferably placed under the control of a functional regulatory sequence in a corn cell, for example in an expression vector according to one of claims 24 or 25. 27. A transformation product obtained from a processed corn plant according to claim 20, a portion of a corn plant according to claim 21, or from a corn cell transformed according to one of claims 19 or 26, characterized in that it has improved digestibility characteristics and that it has a modified ratio between the units S and lignin units G and a modified lignin content. 28. Transformation product according to claim 27, characterized in that the ratio between the units S and the units G is less than 40/60. 29. Transformation product according to one of claims 27 and 28, characterized in that it is fodder. 30. Purified lignin obtained from a maize plant processed according to claim 20, a portion of a maize plant according to claim 21, or a maize cell according to one of claims 19 or 26. , characterized in that it presents in particular a modified ratio between the units S and the units G.  31. purified lignin according to claim 30, characterized in that the ratio between the units S and G units is less than 40/60.